Hematologic Neoplastic Diseases
Hematologic neoplastic diseases of the blood and bone marrow are broadly categorized into acute/chronic leukemias, myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS) (Vardiman J W 2008).
Acute/chronic leukemias are subdivided into myelogenous leukemia and lymphocytic leukemia.
Myeloproliferative neoplasms are subdivided into chronic myelogenous leukemia (CML), polycythemia vera (P. vera), essential thrombocythemia (ET), primary myelofibrosis (PMF), and others.
Myelodysplastic syndromes (MDS) are subdivided into refractory cytopenia with unilineage dysplasia (RCUD), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts (RAEB-1 is defined as having 5-10% myeloblasts and RAEB-2 is defined as having 11-19% myeloblasts in the bone marrow) (Brunning R D 2008).
Explanation of Myelodysplastic Syndromes, Prevalence
Myelodysplastic syndromes are acquired, rare, incurable hematologic diseases characterized by inefficient hematopoiesis due to progressive pancytopenia and abnormal cell differentiation/maturation. These diseases often show chronic propagation over several years and transform into acute leukemia. For these reasons, myelodysplastic syndromes are sometimes called preleukemias.
Myelodysplastic syndromes are primary tumors of bone marrow and their incidence is higher than that of general leukemias. However, the incidence of myelodysplastic syndromes is currently underestimated and it is estimated that many patients are not diagnosed as having myelodysplastic syndromes. Based on the statistical data from the National Health Insurance Corporation, Republic of Korea, 1,845 domestic patients suffered from myelodysplastic syndromes in 2005 and 500-600 new patients are diagnosed as myelodysplastic syndromes annually in Korea. In the United States, the incidence of myelodysplastic syndromes is 1 per 500 individuals over the age of 60 (Newman, Maness-Harris et al. 2012) and 15,000 new patients with myelodysplastic syndromes are found annually and the number is estimated to rise sharply (Barzi and Sekeres 2010).
Diagnostic Methods, Prognosis
Myelodysplastic syndromes are diagnosed based on peripheral blood or bone marrow examination. The degrees of dysplasia are evaluated based on three classifications of hemocytoblasts in the bone marrow, i.e. three different lineages of myeloid cells, erythroid cells, and platelet-forming megakaryocytes, to determine their range and severity. Patients with myelodysplastic syndromes survive for an average of only 22 months, which is similar to the median survival of lung cancer patients, and have a poor 4-5.7-year prognosis. Refractory cytopenia with unilineage dysplasia (RCUD) and refractory anemia with ring sideroblasts (RARS) belong to low risk groups with relatively good prognosis. In contrast, refractory cytopenia with multilineage dysplasia (RCMD) and refractory anemia with excess blasts (RAEB-1 and RAEB-2) that progress to acute leukemia within 9-30 months or reach death belong to groups with poor prognosis. 25% of cases of RAEB-1 and 33% of cases of RAEB-2 progress to AML. Indicators capable of continuously predicting the conditions of patients with these diseases have never been, to our knowledge, reported to date.
Current Therapies for Myelodysplastic Syndromes
At present, there are no effective therapeutic agents for myelodysplastic syndromes. For this reason, patients at the initial stage of the diseases are receiving no suitable treatment for their diseases from the hospital. When the diseases become worse, the patients receive traditional supportive care. Anticancer chemotherapy and allogenic hematopoietic stem cell transplantation are performed in the patients at higher risk.
Over the past 5 years, new concepts of epigenetics have led to an improvement in viability and are thus considered new therapies capable of replacing the best supportive care, which has been a therapeutic principle until now. Continued efforts have been made to develop combination therapies with various drugs. However, the proportion of patients responding to therapeutic agents for myelodysplastic syndromes does not exceed an average of 10% for even the best therapeutic agent. Thus, there is a need in the near future to develop fundamental therapeutic agents in response to the causes of myelodysplastic syndromes and safe therapeutic agents/auxiliary therapeutic agents that are free from side effects caused by protein irritations instead of hematopoietic stem cell transplantation that is substantially impossible to implement on aged patients.
Under these circumstances, the present inventors have found that the dysplasia of erythroid cells caused by culture of bone marrow cells and ex vivo culture of hematopoietic stem cells from patients with myelodysplastic syndromes can be effectively suppressed by treatment of the culture with a human recombinant gelsolin protein. Based on this finding, the present inventors have investigated the possible use of the plasma or intracellular levels of gelsolin as diagnostic and prognostic markers for hematologic neoplastic diseases, including myelodysplastic syndromes.
Gelsolin
Gelsolin is an actin filament-binding protein and is known to cut or cap actin filaments to regulate actin assembly or disassembly (Gremm and Wegner 2000). Gelsolin exists in two forms, cytoplasmic or plasma. Plasma gelsolin differs from cytoplasmic gelsolin by the addition of 25 amino acids to the N-terminus of the molecule (Chauhan, Ji et al. 2008). Plasma gelsolin is secreted from the cell. Gelsolin is present at a high concentration of 200-250 mg/L in normal plasma (Kwiatkowski 1988). Plasma gelsolin is an actin-scavenging protein. When cellular tissue is injured, plasma gelsolin isolates and removes considerable amounts of G-actin and F-actin released into the plasma to protect the microcirculation (Lee and Galbraith 1992).
Diagnostic Values of Gelsolin in Other Diseases
In recent years, a great deal of research has been conducted on the relationship between plasma gelsolin and disease. Plasma gelsolin is mainly produced in and secreted from muscles (Kwiatkowski 1988) and has been investigated in various diseases, such as burns, trauma, brain ischemia/stroke, and respiratory failure. In some disease groups (acute lung injury, septic shock, trauma, and myonecrosis groups) associated with acute cellular injuries and necrosis, the gelsolin values decrease to about 50% of the normal value (Suhler, Lin et al. 1997), and as a result, defense systems by the actin-scavenger system fail to work, which also fatally affects the prognosis of the patients. Decreased gelsolin expression in various tumors, such as colorectal cancer, gastric cancer, lung cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, and renal cancer, was reported to be closely associated with carcinogenesis (Noske, Denkert et al. 2005).
Diagnostic Values of Gelsolin mRNA Level in Myelodysplastic Syndromes
Little is known about the relevance of gelsolin in myelodysplastic syndrome (MDS) patients. Most previous studies have reported the measurement of decreased plasma gelsolin by ELISA and Western blotting but did not verify the suitability for the detection techniques. The measurement of plasma protein levels in acute or chronic leukemia patients was reported but no report has appeared on an increase or decrease of gelsolin at a molecular level.
The results of gene array in bone marrow mononuclear cells of patients with myelodysplastic syndromes (MDS) and patients with bone marrow disease showed patterns of decreasing gelsolin expression (Qi, Chen et al. 2008, Genetics and Molecular Research). However, the expression of each gene was not identified or verified by PCR based on the array results. Further, the mononuclear cells do not reflect the entire cell situation due to the absence of polymorphonuclear cells, which are found only in platelets and buffy coat. Moreover, the mononuclear cells obtained through long-term manual work with ficoll cannot be practically used in a large quantity in environments for patient diagnosis, making it difficult to use as biomarkers. Bone marrow examination is performed only for initial diagnosis and is not useful for prognosis and follow-up. Furthermore, the role of the mononuclear cells as prognostic markers upon follow-up and diagnosis of patients with hematologic tumors remains unknown.
The presence of gelsolin at a low concentration in the bone marrow and peripheral blood serum of acute myeloblastic leukemia patients was confirmed by protein quantitative analysis based on 2-dimensional gel electrophoresis (2DE) (Braoudaki, Lambrou et al. 2013). However, the reasons for an increase in mRNA signal in response to a certain intracellular demand for gelsolin production, the proportion of intracytoplasinic gelsolin or plasma gelsolin produced, and the degradation of secreted gelsolin into extracellular fluids (blood plasma) are not fully understood, making it difficult to predict the correlation between mRNA and plasma gelsolin level.
Most of the current papers on plasma gelsolin in patient groups by ELISA and Western blotting techniques fail to exclude hemolysis of blood samples and overlook the release of intracellular gelsolin into plasma upon hemolysis of samples, making it impossible to measure the exact amount of gelsolin. Also in the case where mononuclear cells are isolated from whole blood samples, a considerable amount of RBCs and platelets are difficult to remove from mononuclear cells. As a result, it is impossible to exclude the effect of gelsolin present at a high concentration in the platelets. No study about gelsolin levels has been reported in sample with erythrocytes and platelets included. There has been no report checking plasma gelsolin level affected by hemolysis in blood.
The present inventors have investigated gelsolin gene expression in bone marrow and peripheral blood of patients with hematologic neoplastic diseases in order to determine the possible use of gelsolin as a factor for the diagnosis and progression of the diseases or a prognostic factor for the diseases. To this end, the present inventors have conducted various experiments to determine whether there are problems in gelsolin expression in patients with myelodysplastic syndromes and various hematologic neoplastic diseases and whether to use the levels of intracytoplasmic protein and mRNA in bone marrow and peripheral blood samples as markers for hematologic neoplastic diseases through various experiments.
Papers and patent publications are referenced and cited throughout the specification, the disclosure of which is incorporated herein by reference in its entirety in order to more clearly disclose the invention and the state of the art to which the invention pertains.
The present inventors have earnestly and intensively conducted research to develop a method for diagnosing hematologic diseases and a method for analyzing the prognosis of hematologic diseases using novel diagnostic markers that are not affected by the levels of platelets and the hemolysis of erythrocytes in subjects, and as a result, have succeeded in diagnosing hematologic diseases and analyzing the prognosis of the diseases by measuring the levels of gelsolin mRNA in peripheral blood or bone marrow from the subjects, accomplishing the present invention.
Therefore, it is one object of the present invention to provide a method for screening a risk group of a hematologic disease.
It is another further object of the present invention to provide a method for analyzing the prognosis of a hematologic disease.
Other objects and advantages of the invention will become more apparent from the following detailed description, claims, and drawings.
According to one aspect of the present invention, there is provided a method for screening a risk group of a hematologic disease, including (a) providing buffy coat of peripheral blood or a bone marrow aspirate isolated from a subject and (b) measuring the expression level of gelsolin mRNA in the buffy coat as a marker for a hematologic disease wherein when the expression level of gelsolin mRNA is measured to be as low as 80% or less or as high as 120% or more of that in a normal group, the subject is diagnosed as being at risk of the hematologic disease.
The present inventors have earnestly and intensively conducted research to develop a method for diagnosing hematologic diseases and a method for analyzing the prognosis of hematologic diseases using novel diagnostic markers that are not affected by the levels of platelets and the homolysis of erythrocytes in subjects, and as a result, have succeeded in diagnosing hematologic diseases, including aplastic anemia and hematologic neoplastic diseases, and analyzing the prognosis of the diseases by measuring the levels of gelsolin mRNA in peripheral blood or bone marrow from the subjects, accomplishing the present invention.
The individual steps of the method will be explained in detail.
(a) Provision of Buffy Coat of Peripheral Blood or a Bone Marrow Aspirate Isolated from a Subject
The diagnostic method of the present invention uses buffy coat of peripheral blood or a bone marrow aspirate isolated from a subject. As used herein, the term “peripheral blood” means blood that circulates systematically and can be collected through the skin. As used herein, the term “bone marrow aspirate” is a soft tissue located in the inner cavity of the bone and refers to a biological sample extracted from bone marrow as a hematopoietic organ by a suitable aspiration method known in the art. For convenience, the terms “bone marrow aspirate” and “bone marrow” are used interchangeably herein. The peripheral blood or bone marrow aspirate is isolated from a subject before use. The bone marrow aspirate is preferably used for initial diagnosis but its isolation from a subject may be limited for follow-up. The use of the peripheral blood isolated from a subject is preferred as a diagnostic sample but this should not be construed as limiting the use of the bone marrow aspirate. The “buffy coat” collected from the peripheral blood and bone marrow aspirate is a white, stripe-shaped layer located between the erythrocyte layer and the plasma layer isolated by concentration gradient centrifugation. The buffy coat essentially contains leukocytes and platelets and may further contain polymorphonuclear cells. The term “gelsolin” refers to an actin-binding protein that is known as a key regulator of actin filament assembly. The screening method of the present invention uses the level of gelsolin mRNA in the buffy coat of the subject.
(b) Measurement of the Expression Level of Gelsolin mRNA in the Buffy Coat as a Marker for a Hematologic Disease
The present inventors have succeeded in demonstrating the correlation between the level of gelsolin mRNA in the buffy coat and the condition of a hematologic disease and the relevance of the profile of gelsolin mRNA levels to the relapse of the hematologic disease. Any known or future method may be used without particular limitation to measure the level of gelsolin mRNA in blood. The present invention is characterized in that the level of gelsolin mRNA in the buffy coat is used as a diagnostic marker for the hematologic disease. There is no restriction on the method for the measurement of mRNA level.
In one embodiment of the present invention, the expression level is measured by a technique selected from the group consisting of quantitative real-time PCR (qPCR), reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE-PCR), multiplex RT-PCR, Northern blotting, nuclease protection assays, in situ hybridization, serial analysis of gene expression (SAGE), RNA microarray, RNA microarray and gene chips, and RNA sequencing (RNA-seq). Specifically, the level of gelsolin mRNA in the buffy coat may be measured by qPCR in accordance with a suitable method known in the art. qPCR was used in the Examples section that follows.
In one embodiment of the present invention, the hematologic disease is aplastic anemia or a hematologic neoplastic disease. More specifically, the hematologic neoplastic disease is selected from the group consisting of acute leukemias, chronic leukemias, myeloproliferative neoplasms, and myelodysplastic syndromes. The average gelsolin mRNA levels of biological samples (peripheral blood and/or bone marrow samples) obtained from patients with the above diseases are lower than those of healthy subjects, as confirmed by the present inventors.
The acute leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia. The myeloproliferative neoplasms include, but are not limited to, chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The myelodysplastic syndromes include, but are not limited to, refractory cytopenia with unilineage dysplasia, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts-1 (RAEB-1), and refractory anemia with excess blasts-2 (RAEB-2).
According to the method of the present invention, when the expression level of gelsolin mRNA in the buffy coat is measured to be 80% or less of that in a normal group, the subject is diagnosed as being at risk of the hematologic disease. Meanwhile, when the expression level of gelsolin mRNA in the buffy coat is measured to be 120% or more of that in a normal group, the subject is diagnosed as being at risk of a hematologic neoplastic disease just before or immediately after progression to other diseases.
More specifically, the expression level of gelsolin mRNA in the buffy coat of the subject diagnosed as having the hematologic disease is 80% or less, 70% or less, 60% or less, 50% or less, 40% or less or 30% or less of that in a healthy subject. The lower expression level means the higher possibility that the subject will be diagnosed as having the hematologic disease. Particularly, the expression level of mRNA gelsolin in a leukemia patient is, on average, lower than those in myelodysplastic syndrome and myeloproliferative neoplasm patients.
In one embodiment of the present invention, the subject may be a bone marrow dysplasia patient. In this embodiment, when the mRNA expression level of gelsolin in the subject is measured to be 120% or more of that in a normal group, the bone marrow dysplasia is predicted to be progressed to a pre-leukemic stage.
In one embodiment of the present invention, the pre-leukemic stage is refractory anemia with excess blasts-2 (RAEB-2). The RAEB-2 is interpreted to include acute myelogenous leukemia (AML) immediately after development from RAEB-2.
The levels of gelsolin in most risk groups of hematologic diseases are measured to be 80% or less of those in normal groups. Exceptionally, higher gelsolin mRNA values are observed in patients during progression from a disease to another, for example, from myelodysplastic syndrome (MDS) to acute myelogenous leukemia (AML), than those observed in healthy subjects.
More specifically, the levels of gelsolin mRNA in buffy coats of patients with hematologic neoplastic diseases just before or immediately after progression to other diseases are measured to be 120% or more, 130% or more, 140% or more or 150% or more of those in healthy subjects. High levels of gelsolin mRNA are diagnostic features of patients during progression from myelodysplastic syndrome (MDS) to acute myelogenous leukemia (AML), as mentioned earlier.
In one embodiment of the present invention, the method is not affected by the level of platelets and the hemolysis of erythrocytes in the sample. Attempts have been made to use plasma gelsolin levels for disease diagnosis. However, none of the attempts were successful in obtaining consistent results because the levels of gelsolin in plasma are greatly affected by various factors, such as platelet level and erythrocyte hemolysis in samples, and sufficiently reflect the conditions of diseases, making it impossible to use the levels of gelsolin in plasma for disease diagnosis. The present inventors have found that when buffy coat isolated from peripheral blood or bone marrow is used, consistent results can be obtained irrespective of the level of platelets and the hemolysis of erythrocytes in samples. Based on this finding, the present inventors have succeeded in finding a method for the diagnosis of a hematologic neoplastic disease.
According to a further aspect of the present invention, there is provided a method for analyzing the prognosis of a hematologic neoplastic disease, including (a) measuring a first expression level of gelsolin mRNA in buffy coat of peripheral blood or a bone marrow aspirate isolated from a subject and (b) measuring a second expression level of gelsolin mRNA in another buffy coat of peripheral blood or a bone marrow aspirate isolated from the subject after the lapse of time when the condition of a disease is expected to be ameliorated and comparing the first and second expression levels wherein when the second expression level is at least 3 times higher than the first expression level, the disease is considered to be relapsed.
The present inventors have demonstrated that the current condition of a disease can be diagnosed from the expression level of gelsolin mRNA measured upon initial diagnosis of a subject and the prognosis of a disease can be analyzed through observation of the relative profile of gelsolin mRNA expression levels upon follow-up of a subject. The individual steps of the method will be explained in detail.
(a) Measurement of a First Expression Level of Gelsolin mRNA in Buffy Coat of Peripheral Blood or a Bone Marrow Aspirate Isolated from a Subject
The analytical method and the diagnostic method share the use of the level of gelsolin mRNA in buffy coat in common. The same description of the diagnostic method is applicable to the analytical method and is thus omitted to avoid duplication.
The analytical method of the present invention uses two buffy coat samples isolated at different time points from a subject. As used herein, the term “first expression level” means the expression level of gelsolin mRNA in the buffy coat sample isolated earlier from a subject. The first and second expression levels of gelsolin mRNA can be represented as relative values based on the expression level of gelsolin mRNA in a healthy subject.
The first expression level can be measured by the same technique described in the diagnostic method.
(b) Measurement of a Second Expression Level of Gelsolin mRNA in Another Buffy Coat of Peripheral Blood or a Bone Marrow Aspirate Isolated from the Subject after the Lapse of Time when the Condition of a Disease is Expected to be Ameliorated and Comparison of the First and Second Expression Levels
The analytical method of the present invention is to predict and prospect a change of a disease. The analytical method of the present invention optionally includes ameliorating the condition of a disease by proper treatment after measurement of the first expression level and following-up the therapeutic effect and the change of the condition. The time when the condition of a disease is expected to be ameliorated means the time taken until the condition of the disease is generally expected to be ameliorated after proper treatment and may vary depending on complex factors, such as general health of the subject, type of the treatment, and sensitivity to the treatment. The expression “time when the condition of a disease is expected to be ameliorated” considers the fact that a significant difference between the first and second expression levels is generally difficult to expect when the second expression level is measured in too short a time after measurement of the first expression level but is not necessarily premised on the amelioration of the condition. It may take several hours or even several days depending upon the kind of the disease or the therapeutic method.
Thereafter, the first expression level is compared with the second expression level. When the second expression level is about 3 times or more, 4 times or more, 5 times or more or 6 times or more higher than the first expression level, the disease is judged to be relapsed.
When the second expression level is almost the same as or lower than the first expression level, the disease is determined to respond to the treatment or to progress slowly. When the second expression level increases up to about 3 times the first expression level, the disease is not judged to be relapsed. However, when the second expression level is 5 times or more the first expression level, the disease is judged to be relapsed. In this case, for example, refractory cytopenia with multilineage dysplasia (RCMD) is judged to be relapsed into refractory anemia with excess blasts-1 (RAEB-1) or RAEB-1 is judged to be relapsed into acute myelogenous leukemia (AML) (see FIG. 3).
In one embodiment of the present invention, the buffy coat samples used in steps (a) and (b) are isolated from peripheral blood. Alternatively, the buffy coat samples may be isolated from bone marrow. However, the use of the buffy coat samples isolated from peripheral blood is preferred for the convenience of follow-up.
In one embodiment of the present invention, the expression levels are measured by a technique selected from the group consisting of quantitative real-time PCR (qPCR), reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE-PCR), multiplex RT-PCR, Northern blotting, nuclease protection assays, in situ hybridization, serial analysis of gene expression (SAGE), RNA microarray, RNA microarray and gene chips, and RNA sequencing (RNA-seq). Specifically, the expression levels of gelsolin mRNA may be measured by the selected technique in accordance with a suitable method known in the art.
In one embodiment of the present invention, the hematologic disease is aplastic anemia or a hematologic neoplastic disease. More specifically, the hematologic neoplastic disease is selected from the group consisting of acute leukemias, chronic leukemias, myeloproliferative neoplasms, and myelodysplastic syndromes. The present inventors have demonstrated the correlation between the relapse profiles of the hematologic neoplastic diseases and the profiles of gelsolin mRNA.
According to the diagnostic method and the analytical method of the present invention, physical and economic burdens on patients can be reduced and consistent diagnostic and analytical results can be obtained without being affected by other ambient factors.
The features and advantages of the present invention are summarized as follows:
(a) the present invention is effective in screening a risk group of a hematologic disease;
(b) the present invention is effective in analyzing the prognosis of a hematologic disease;
(c) the use of the present invention enables consistent screening of a risk group of a hematologic disease while minimizing the influence of ambient factors; and (d) the use of the present invention enables the analysis of prognosis of a hematologic disease in a more accurate manner.